Vol. 8(37), pp. 3422-3434, 10 September, 2014 DOI: 10.5897/AJMR2014.6802 Article Number: B5A96EE47927 ISSN 1996-0808 African Journal of Microbiology Research Copyright © 2014 Author(s) retain the copyrighht of this article http://www.academicjournals.org/AJMR Full Length Research Paper Morphological, physiological and pathogenic variability of small-spore Alternaria sp. causinng leaf blight of Solanaceous plants in Allgeria BESSADAT Nabahat1*, SIMONEAU Philippe2, BENICHOU Soumaya1, SETTI Benali3, Kihal Mabrouk1 and HENNI Djamel Eddine1 1Laboratoire de Microbiologie Appliquée, Université d’Oran Es-Senia, BP15224 El M’naouer 31000 Oran, Algeria. 2SFR QUASAV 4207, UMR 1345 IRHS, Université d’Angers, 2 boulevard Lavoisier, France. 3Institut des Sciences Agronomiques, Université de Chlef, Algeria. Received 25 February, 2014; Acceppted 9 June, 2014 Due to premature defoliation, early blight epidemics can cause major yield losses. Large-spore Alternaria species such as A. solani and A. tomatophila have long been recognized as important pathogens responsible for such blight disease in the famiily Solanaceeae and thus represent a serious risk for crop production. Small-spore Alternaria species have also been frequently isolated from plant samples with typical blight symptoms but their incidence as primary pathogens is often controversial. In order to study the diversity of small-spore Alternaria species, 32 isolates were selected from a larger collection of 130 isolates from infected leaves, fruits and sttems of tomato from various growing regions of North-West Algeria. Morphological characterization under standard conditions annd polymerase chain reaction (PCR) analyses using specific primers to amplify a part of the ITS regions and the 5.8S gene were conducted to confirm their identification as members of the altternata section. They were then examined according to morphological characteristics of conidia and sporulation patterns on potato carrot agar (PCA) and were segregated into three morphological speccies: A. alternata, A. tenuissima and A. arborescens. Colony type, substrate colour, margin, zonation, pigmentation, colony diameter and conidia production were studied on potato sucrose agar (PSA). Physiological parameters and nutritional requirements of the isolates were also assessed and a data matrix based on cluster analysis and Euclidean distance was constructed. Results of pathogenicity test on tomato showed obvious diversity among the isolates and they could be separated into two groups based on their virulence. The dendrogram based on the influence of cultural, nutritionnal and physiological characters suggests moderate heterogeneity within the populations of A. alternata and A. tenuissima. The small-spore species formed five clusters that fundamentally paralleled the morphological groupings. However, the results provided no evidence for geographical and pathogenicity clustering of isolaates. Key words: Epidemiology, Solanaceae, small-spore Alternaria species, pathogenic variability, cultural characters, physiological diversity. INTRODUCTION Tomato (Lycopersicon esculentum L) often called as important vegettable crops grown in the world. It is “queen of kitchen” is one of the oldest known and commonly used for culinary purposes and consumed in Bessadat et al. 3423 diverse ways, including raw, as an ingredient in many growth and sporulation of fungi are temperature, hydro- dishes, sauces, salads, and drinks. In Algeria, it repre- genion concentration and both carbon and nitrogen sents a surface of 12173 Ha which covers 57.36% of the sources. A small variation in these factors may induce global cultivated area. Its production is ca 10 MQx of clear differences in their morphological characters, which 3.8 MQx is for industrial use (that is equivalent to growth and sporulation (Bilgrami and Verma, 1978). Con- 95% of the total industrial culture production) (Snoussi, cerning pathogenic variability, it has been well establish- 2009). Due to its high adaptability, early blight has the hed that A. solani and A. tomatophila could be con- potential to become a serious threat for tomato crops in sidered as primary pathogens responsible for early blight northwestern Algeria, because of the favorable environ- of tomato (Simmons, 2000; Lourenço et al., 2009), while ment; it affects foliage as well as tomato fruit reducing tomato isolates belonging to the alternata section have yield both qualitatively and quantitatively. Epidemics often been considered as successful invaders of pre- occur when the weather is warm and dry with short formed necrotic lesions with poor capability to infect periods of high moisture and moderate temperatures (18 green foliage in the field (Spits et al., 2005). Despite this, to 30°C). The fungal pathogens responsible for the it is also well recognized that pathogenic populations disease belong to the genus Alternaria and infect the (pathotypes) with narrow host range exist within the plant through conidia which are either wind-blown or alternata section. A. arborescens responsible for the to- splashed onto plant surfaces (Andersen and Frisvad, mato stem canker constitutes a typical example (Grogan 2004; Leiminger et al., 2010). Both large-spore forming et al., 1975, Mesbah et al., 2000). species (A. solani and A. tomatophila) and small-spore The objective of this study was to evaluate the impor- Alternaria spp. have been isolated from diseased tomato tance of small–spore Alternaria species and to analyze (Simmons, 2000). the phenotypic variability within these populations iso- The Alternaria genus includes nearly 275 species lated from tomato with early blight symptoms. We have (Simmons, 2007) with saprophytic and plant-pathogenic therefore collected several isolates from diseased tomato lifestyles that may affect crops in the field or cause har- in Algeria, analyzed their morphological characteristics vest and post-harvest decay of plant products (Logrieco based on growth and sporulation patterns and cultural et al., 2009). Alternaria taxonomy has long been mainly behaviour on different media. We have also studied their based on conidia morphology and sporulation pattern. virulence on tomato and showed that they can be divided Nishimura and Kohmoto (1983), using a statistical ana- into two groups based on their relative aggressiveness. lysis of the size of conidia concluded that Alternaria iso- To the best of our knowledge, this constitutes the first lates producing small spores belong to what they called a report of leaf blight in solanaceous crops caused by "collective species" alternata. From then, several authors small-spore Alternaria that is belonging to the alternata (Kusaba and Tsuge, 1995; Johnson et al., 2000; Tsuge, section, in the northwestern Algeria. 2003) have accepted these findings. To facilitate segre- gation and identification, Simmons and Roberts (1993) introduced the 3-dimensional sporulation pattern as a MATERIALS AND METHODS means of scoring small-spore isolates from Asian pears into groups. They described six major groups with cha- Isolation racteristic sporulation patterns of which three will be Samples of fresh infected leaf, stem and fruit of tomato were collec- treated in this paper. More recently based on phylogene- ted from 65 fields of commercial farmers located in the main vege- tic studies, the Alternaria genus was separated into 24 table producing cities in northwestern of Algeria including: Oran, sections and small-spore forming Alternaria were Mascara, Mostaganem, Ain témouchent Relizane, Tlemecen and grouped into the alternata section that comprises almost Sidi Bel-Abbès. 60 Alternaria species (Woudenberg et al., 2013; Lawrence et al., 2013). The molecular variation within the Identification of isolates alternata section is low and these species were thus mainly differentiated based on phenotypic variation. Isolation of Alternaria strains was done from small pieces of in- Phenotypic variations within fungal populations can fected tissues after surface disinfection using 3% sodium hypo- generally be detected using morphological, cultural and chlorite (NaOCl) for 5 min. Tissue samples were then rinsed with pathogenic criteria. Understanding pathogen population sterile water and placed onto potato sucrose agar (PSA) medium structure and mechanisms by which such variations arise and incubated at 25°C for 7 days. Cultures that were contaminated with bacteria were transferred on PSA amended with streptomycin within a population is of paramount importance for devi- (50 µg ml-1). Pure cultures were obtained for each of the isolates sing a successful disease management strategy. using the single spore technique according to Hansen (1926). Most important environmental factors controlling the Single spore colony was transferred to the same PSA medium. The *Corresponding author. E-mail: [email protected]. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0International License 3424 Afr. J. Microbiol. Res. Table 1. Scale for evaluation of the mean mycelia growth and conidial production of Alternaria isolates. Mean mycelial growth (mm/day) Conidial production (×105 spores.mm-2) Rating 0 to 3.0 0 to 10 0 3.1 to 6.0 10.1 to 20.0 1 6.1 to ≤ 20.1 to ≤ 2 130 pure isolates were stored at 4°C on PSA slants for further Effect of pH study. Identification of small spore Alternaria was performed accor- ding to a previously established protocol (Simmons, 2007). To test the influence of pH on fungal growth,